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='''Testing 1/100 diluted tamplate from human cell MDA amplicon'''= ==Objective== *Test if 1/100 diluted template of MDA amplcon can perform a better PCR amplification (Better ratio template:primer set or less interference from salts). ==Samples & Materials== *Enzyme - Taq 2X master mix (NEB) *Positive control template - 1/100 diluted Jurkat cell genomic DNA (NEB) -> 1 ng/uL *Human genome primers **18S-306 (cho. 21) - previously tested **2-1 (cho.2, 540 bp, Tm 52C) *Templates (1 uL): A3, A6, A7, B4, B7, C3, C4, C8, D6(p-Pos), D7(p-Neg), Pos(1/100 gDNA = 1 ng/uL), H2O ==Exp. Design== *Templates (1 uL): 1/100 diluted MDA products **A3, A6, A7, B4, B7, C3, C4, C8, D6(p-Pos), D7(p-Neg), Pos(1 ng/uL gDNA), H2O Templates (A->H) --------------------------------------------------------------------------- A3 A6 A7 B4 B7 C3 C4 C8 p-Pos p-Neg Pos H2O --------------------------------------------------------------------------- Primer P1 18S-306 (cho. 21, 306 pb, Tm 59C) P2 2-1 (cho.2, 540 bp, Tm 52C) ==Procedures== *Thaw the Taq2X enzyme and primers. *Dilute all MDA template. Prepare diluted Pos template (1 ng/uL) *Set up PCR program (GENE59, GENE52). *Master Mix - x 3 (Primers are various) **Total reactions: 12 rxns 1 rxn 12+2 rxn --------------------------- H2O 3.5 49.0 Primer 0.5 7.0 Taq 2X 5.0 70.0 --------------------------- 9.0 126.0 (uL) 126/14=9 *Prepare 3 trips of 8-well PCR tubes and transfer the 1 uL template. *Transfer 9 uL of master mix into each reaction *Perform PCR reaction in thermocycler **Strip P1 goes to block A (program GENE59: Tm59, 30 cycles) **Strip P2 and P3 go to block B (program GENE52: Tm52, 30 cycles) *Gel Electrophoresis **1.5% agarose: (1.5 g agarose +100 mL 0.5X TBE + 8 uL SYBR safe -> four small tray with 8-well comb) **Run at 135 V for 20 min. **Loading: Sample 7 uL + 2 uL Blue juice -> 7 uL. Diluted ladder 7 uL ==Results== [[Image:ZhangLab_2 2009-02-28 test2 primer_18S.bmp|400px]] [[Image:ZhangLab_2 2009-02-28 test2 primer_2-1.bmp|400px]] *Left: Test by primer 18S (306 bp) *Right: Test by primer 2-1 (540 bp) ==Result & Discussion== *1/100 dilution didn't give us better PCR amplification. Even the p-Pos didn't show up. *The PCR template using MDA amplicon might be used in a range between 1/1 ~ 1/10 dilution. *Pick up B7 and C8 (from Test 2) for 2nd MDA.
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